16S rRNA AND SOXB GENE SEQUENCE ANALYSIS OF A SULFIDIC SPRING IN BROMIDE, OKLAHOMA

Although numerous metabolic strategies are known to have evolved in sulfide-oxidizing bacteria to utilize sulfur compounds, our knowledge is limited regarding the diversity of sulfide oxidation metabolic genes among different taxonomic groups, and how much nucleotide variation in the metabolic genes can exist and still result in the same function. The hypothesis being tested is that variations in gene sequence, which cause enzyme structural changes, result in optimal enzyme function under specific geochemical conditions for that taxonomic group; if a different group acquired the gene from horizontal gene transfer, then that group would be predisposed to the specific geochemistry optimal for enzyme function. Variations of the soxB gene, of the sulfide oxidation pathway (Sox), were analyzed along a geochemical gradient within a sulfidic spring in Bromide, OK. The H₂S concentration decreased away from the spring orifice from 188 to 0 µmol/L at 14m in the outflow channel, but dissolved O₂ increased from 5.6 to 150 µmol/L, and pH increased from 6.8 to 7.8. Analysis of 16S rRNA genes collected from microbial mat samples upstream (highest H₂S) were dominated by sulfide-oxidizers (Gammaproteobacteria and Epsilonproteobacteria), but there were also sulfide-oxidizers along the outflow channel peripheries where H₂S was <29 µmol/L. soxB genes were retrieved from all sample locations, and homology (although poor) was to various previously described Proteobacteria. Sequence Operational Taxonomic Units (OTUs) differed along the channel, such that 60% of the sequences from the orifice in July and 45% in October clustered into four OTUs found only at the orifice. The middle channel segment was dominated by two OTUs (98% of the sequences in July and 97% in October) that were minor OTUs elsewhere. At the terminus, 60% of the soxB sequences in July and 88% in October clustered into OTUs unique to this segment of the channel. The potential functional importance of significant amino acid sites within the soxB protein was evaluated from evolutionary trace analyses of retrieved soxB genes. Conserved and functionally divergent amino acid residues were identified that indicate altered enzyme efficiency under varying geochemical conditions that is also consistent with phylogenetic diversification along the spring outflow channel.